The long term objectives are to define, in Saccharomyces cerevisiae, the roles of various nucleotide excision repair (NER) protein factors in damage recognition and in the assembly of the NER machinery at the damage site. The proposed studies will determine the role of the newly identified gene RFI1 in the repair of non-transcribed DNA and they will examine the damage binding properties of the purified Rfi1 protein. Genetic and biochemical studies will be performed to determine the role of the C4 and C3HC4 sequence motifs present in the Rfi1 protein and in the Rad16 protein. The physical and functional interaction of Rfi1 with the Rad7-Rad16 protein complex and with the other NER factors will be studied, and the role of the C3HC4 sequence present in Rfi1 and Rad16 on promoting association between these two proteins will be examined. The Abf1 protein is a component of the Rad7-Rad16 complex. The role of ABF1 in the repair of non-transcribed DNA will be examined and the damage binding properties of Abf1 protein will be studied. The Mms19 protein complex, which is comprised of four proteins of 100 (Mms19), 80, 55, and 43 kDa will be studied further. The identity of these Mmsl9 associated proteins will be determined by mass spectrometry, and genetic and biochemical studies will examine the role of these subunits and of the Mmsl9 complex in NER and in Pol II transcription. The order by which different NER factors assemble on damaged DNA will be analyzed by gel retardation and by DNase I footprinting. The role of the Rad7-Rad16 complex and of associated proteins in chromatin remodeling will be analyzed, and the effect of these proteins on the removal of UV lesions from nucleosomal DNA examined in the reconstituted system. Xeroderma pigmentosum (XP) patients are defective in NER of UV damaged DNA, and as a consequence, they suffer from a high incidence of skin cancers. Our studies in yeast should continue to provide important insights on the role of various protein factors in damage recognition and other steps of the NER process in eukaryotes, including in humans.